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The use of biologically active small molecules to perturb biological functions holds enormous potential for investigating complex signaling networks. However, in contrast to animal systems, the search for and application of chemical tools for basic discovery in the plant sciences, generally referred to as “chemical genetics,” has only recently gained momentum. In addition to cultured cells, the well-characterized, small-sized model plant  Arabidopsis thaliana  is suitable for cultivation in microplates, which allows employing diverse cell- or phenotype-based chemical screens. In such screens, a chemical's bioactivity is typically assessed either through scoring its impact on morphological traits or quantifying molecular attributes such as enzyme or reporter activities. Here, we describe a facile forward chemical screening methodology for intact  Arabidopsis  seedlings harboring the β-glucuronidase (GUS) reporter by directly quantifying GUS activity  in situ  with 4-methylumbelliferyl-β-D-glucuronide (4-MUG) as substrate. The quantitative nature of this screening assay has an obvious advantage over the also convenient histochemical GUS staining method, as it allows application of statistical procedures and unbiased hit selection based on threshold values as well as distinction between compounds with strong or weak bioactivity. At the same time, the  in situ  bioassay is very convenient requiring less effort and time for sample handling in comparison to the conventional quantitative  in vitro  GUS assay using 4-MUG, as validated with several  Arabidopsis  lines harboring different GUS reporter constructs. To demonstrate that the developed assays is particularly suitable for large-scale screening projects, we performed a pilot screen for chemical activators or inhibitors of salicylic acid-mediated defense signaling using the  Arabidopsis PR1p::GUS  line. Importantly, the screening methodology provided here can be adopted for any inducible GUS reporter line.

frontiers in plant science

Facile high-throughput forward chemical genetic screening by in situ monitoring of glucuronidase-based reporter gene expression in Arabidopsis thaliana